Natural Antioxidant Boosters for Honey: Effects of Portulaca oleracea and Salicornia perennans Powders and Extracts

Abstract

Honey is a valuable carrier for phytochemicals, yet data on halophyte-based fortification remain scarce. This study evaluated the impact of Portulaca oleracea and Salicornia perennans, applied as powders 1.5–4.5% or ethanol extracts 1.2–1.8%, on the antioxidant and functional properties of rapeseed and multifloral honeys. Antioxidant capacity (DPPH, ABTS), total phenolic (TPC), and flavonoid contents (TFC) increased significantly in a concentration-dependent manner, with powders consistently outperforming extracts. The strongest effects were obtained with 4.5% powders, yielding up to 77.83% DPPH, 99.11% ABTS, 14.85 mg gallic acid equivalents (GAE) per 100 g TPC, and 44.15 mg QE/100 g TFC-values surpassing controls and synthetic standards. Colorimetric and oxidative stability assays confirmed that enriched honeys exhibited slower browning and reduced peroxide/TBARS accumulation during storage. Sensory analysis further indicated improved color, aroma, taste, and overall acceptability. Between species, Salicornia showed slightly stronger effects than Portulaca, while multifloral honey provided greater synergy than rapeseed. These results demonstrate that halophyte powders are effective natural enhancers of honey’s antioxidant, technological, and sensory qualities, supporting their use in functional food development.

1. Introduction

Honey is a natural bee product synthesized from floral nectar by Apis mellifera and has been consumed since antiquity as both a food and a medicine [1]. Its composition is dominated by simple carbohydrates—mainly glucose and fructose—which account for about 80% of solids [2]. In addition to sugars, honey contains 14–20% water, proteins, free amino acids (especially proline), vitamins, minerals, organic acids, and numerous bioactive compounds [3,4]. The balance of these components, combined with low water activity, contributes to honey’s remarkable stability [5]. Among its biologically active constituents, antioxidants play a key role. These include enzymatic antioxidants (catalase, peroxidase) and non-enzymatic molecules such as phenolic acids, flavonoids, carotenoids, and vitamins [6,7]. Concentrations of phenolic compounds in some honey varieties exceed 250 µg·g⁻¹, contributing significantly to their antimicrobial and radical-scavenging activity [8,9]. The antioxidant and antimicrobial properties of honey form the basis of its therapeutic potential, which includes wound healing, immune modulation, and gastrointestinal protection [10,11].

The bioactivity of honey varies significantly depending on floral origin, geographical region, and processing conditions [12,13]. To further enhance its functional potential, researchers have explored enrichment with natural plant ingredients [14]. Honey serves as an ideal carrier for bioactive compounds—its high sugar concentration stabilizes phytochemicals, and its pleasant taste masks the bitterness of certain plant extracts [15]. Previous studies have shown that fortification with herbs, spices, or fruit extracts (e.g., lavender, nettle, ginger, or elderberry) can increase radical-scavenging capacity several-fold compared with untreated honey [16,17,18]. However, most plant additives originate from non-saline environments, whereas halophytes—plants naturally adapted to high salinity—accumulate osmoprotectants and stress-induced antioxidants with a distinctive biochemical composition. Two halophytes, purslane (Portulaca oleracea) and glasswort (Salicornia perennans), are particularly promising yet underexplored candidates for honey fortification [19,20,21,22]. Their tissues are enriched with ferulic, caffeic, and p-coumaric acids, quercetin derivatives, and mineral antioxidants (Mg, K, Zn), which arise from metabolic adaptation to oxidative stress in saline soils [23,24,25,26,27,28]. This unique phytochemical profile differentiates halophytes from conventional herbs or fruit materials previously used in honey enrichment.

The antioxidative mechanism of phenolic acids and flavonoids is based on electron or hydrogen atom donation to neutralize free radicals, chelation of transition metals (Fe²⁺, Cu²⁺) to prevent Fenton-type reactions, and termination of lipid peroxidation chains through the stabilization of reactive intermediates [29,30,31]. Considering these mechanisms, halophyte-derived antioxidants could synergistically enhance honey’s oxidative stability and nutritional quality. We hypothesized that halophyte powders would outperform ethanol extracts due to their higher content of bound phenolics and fiber-associated antioxidants, which provide gradual release and prolonged radical-neutralizing effects. Moreover, fortifying honey with P. oleracea and S. perennans aligns with the principles of sustainable apiculture and circular bioeconomy, offering opportunities for the valorization of arid-zone flora and the development of natural, high-value functional products.

Therefore, the present study aimed to evaluate the effects of P. oleracea and S. perennans, applied as dried powders and ethanol extracts at different concentrations, on the antioxidant potential, total phenolic content, and flavonoid content of rapeseed and multifloral honeys. Antioxidant activity was determined using DPPH and ABTS assays, while spectrophotometric methods were applied for quantifying total phenolics and flavonoids. The results are expected to elucidate the role of halophyte bioactives in enhancing the health-promoting properties of honey and to contribute to the broader development of functional bee products within sustainable food systems.

2. Materials and Methods

2.1. Plant Material

Fresh aerial parts of P. oleracea L. (purslane) and S. perennans Willd. (glasswort) were collected during the active vegetation period (June–July 2025) in the Kyzylorda region of Kazakhstan, which is characterized by an arid climate and saline soils. These environmental conditions are known to stimulate the biosynthesis of phenolic compounds in halophytic plants. After manual cleaning to remove soil and impurities, the samples were air-dried at 25 ± 2 °C in a shaded, ventilated room until constant weight was achieved. The dried material (residual moisture 6–8%) was milled using a laboratory grinder (IKA A11 Basic, Staufen, Germany) and passed through a 0.5 mm sieve. Voucher specimens were authenticated and stored in the departmental herbarium (codes PO/7/2025 and SP/6/2025). The composition of honey samples enriched with powders and extracts of P. oleracea and S. perennans is presented in

Table 1. Ingredients used to prepare the honey samples enriched with P. oleracea and S. perennans.

Form of Enrichment	Honey (g)	Plant Powder (g)	Extract (mL)	Ethanol 70% (mL)	Final Concentration (% w/w, of Honey Mass)
Control (none)	100.0	-	-	-	-
Powder 1.5%	98.5	1.5	-	-	1.5
Powder 3.0%	97.0	3.0	-	-	3.0
Powder 4.5%	95.5	4.5	-	-	4.5
Extract 1.2%	98.8	-	0.6	0.6	1.2
Extract 1.5%	98.5	-	0.75	0.75	1.5
Extract 1.8%	98.2	-	0.9	0.9	1.8

. An illustrative schematic diagram summarizing the entire workflow—from plant collection → extract/powder preparation → honey fortification → analytical and sensory testing—has been added to the Supplementary Information as Figure S1 for clarity and better visualization of the experimental design.

2.2. Preparation of Plant Extracts

To obtain ethanol extracts, 20 g of the dried and milled plant material was suspended in 200 mL of 70% aqueous ethanol, maintaining a solid-to-liquid ratio of 1:10 (w/v). The mixture was first sonicated at 40 °C for 1 h to accelerate solvent penetration into plant tissues and then gently agitated on a laboratory shaker (150 rpm, 30 min) to ensure thorough extraction. The suspension was subsequently filtered through standard laboratory paper and clarified by centrifugation at 3500× g for 20 min. The resulting supernatant was concentrated under reduced pressure at 40 °C until most of the solvent was removed, producing a viscous extract. This residue was dissolved in 10 mL of 50% ethanol to yield stock solutions, which were stored in amber vials at 4 °C until use. In parallel, a portion of the original dried powders was kept without solvent treatment and directly applied in honey fortification experiments.

2.3. Honey Samples and Experimental Design

Two commercial honeys from certified apiaries in the Akmola region of Kazakhstan were used: (i) rapeseed (Brassica napus) and (ii) multifloral honey collected from mixed wildflower sources. Both honeys were filtered to remove debris but otherwise unprocessed. Before enrichment, both rapeseed and multifloral honeys were analyzed for their basic physicochemical parameters to verify authenticity and quality. The analyses included measurements of moisture content, pH, electrical conductivity, free acidity, reducing sugars, sucrose, and hydroxymethylfurfural (HMF). All determinations were carried out in triplicate according to AOAC [32] standard. The results are summarized in

Table 2. Basic physicochemical properties of the rapeseed and multifloral honeys used in this study.

Parameter	Unit	Rapeseed Honey	Multifloral Honey
Moisture content	%	17.4 ± 0.2	17.9 ± 0.3
pH	–	3.76 ± 0.05	3.64 ± 0.04
Free acidity	meq/kg	24.5 ± 1.3	27.2 ± 1.5
Electrical conductivity	mS/cm	0.29 ± 0.02	0.46 ± 0.03
Reducing sugars (glucose + fructose)	%	72.6 ± 0.8	71.8 ± 0.7
Sucrose	%	2.1 ± 0.2	2.4 ± 0.2
Hydroxymethylfurfural (HMF)	mg/kg	9.8 ± 0.6	11.2 ± 0.7

.

Both honeys met all international quality criteria, exhibiting moisture content below 18%, moderate acidity, and low HMF values, confirming freshness and absence of thermal degradation. The slightly higher electrical conductivity of multifloral honey reflects its diverse floral origin and higher mineral content compared with rapeseed honey. These characteristics indicate that both honey types were suitable for subsequent fortification and comparative antioxidant analysis. All concentrations are expressed as % w/w relative to the honey mass (i.e., 1.5–4.5% of total honey weight). Specifically, powders were added at levels of 1.5%, 3.0%, and 4.5% (w/w), while ethanol extracts were added at 1.2%, 1.5%, and 1.8% (v/w, adjusted with 70% ethanol). Control samples consisted of untreated honeys. Each formulation was prepared in triplicate, homogenized by manual stirring followed by vortexing (2 min), and stored in sterile glass jars (250 mL) at 21 ± 1 °C in the dark for 90 days. During storage, samples were gently mixed every 30 days. For stability and sensory tests, the concentration of 3% powder was selected as representative, as this level provided both high antioxidant enhancement and acceptable sensory properties in preliminary trials.

All honey samples (control and enriched) were stored for 90 days at 21 ± 1 °C in the dark, protected from direct sunlight and moisture, in airtight sterile glass jars 250 mL. The jars were kept in a temperature-controlled laboratory cabinet with limited air exchange to minimize oxidation and evaporation. To ensure uniformity, samples were gently mixed every 30 days using a sterile glass rod. Stability parameters—including color (Section 2.7), oxidative indicators (Section 2.9), and sensory quality (Section 2.10)—were determined at 0, 30, and 90 days of storage.

2.4. Sample Preparation for Analytical Assays

Prior to analysis, crystallized honeys were liquefied in an ultrasonic bath at 40 °C for 60 min. For chemical assays, 5 g of each honey variant was dissolved in 20 mL distilled water containing 1% acetic acid and subjected to sonication (30 min, 40 °C). After filtration through 0.45 µm membranes (Sartorius, Göttingen, Germany), the filtrates were evaporated under vacuum, reconstituted in 15 mL of 1% acetic acid, and further purified on Sep-Pak C18 cartridges. Bound polyphenols were eluted with 10 mL methanol, evaporated, and re-dissolved in 2.5 mL MS-grade methanol. Final solutions were filtered through 0.22 µm nylon filters before instrumental analysis.

2.5. Determination of Antioxidant Activity

DPPH radical scavenging activity was determined according to the method of Brand-Williams et al. [33] with minor modifications. A 0.1 mM solution of DPPH in methanol was prepared and adjusted to an absorbance of 0.95 ± 0.03 at 517 nm. To each well of a 96-well microplate, 10 µL of honey extract was added to 140 µL of DPPH solution. Plates were incubated in the dark for 30 min at room temperature, and absorbance was measured at 517 nm (SpectraMax M5, Molecular Devices, San Jose, CA, USA).

ABTS radical scavenging activity was evaluated following the procedure [34]. The ABTS⁺ radical was generated by incubating 7.5 mM ABTS with 2.5 mM potassium persulfate in the dark for 16 h. The working solution was diluted with methanol to achieve an absorbance of 0.90 ± 0.02 at 734 nm. For analysis, 10 µL of sample was mixed with 140 µL of ABTS⁺ solution, and absorbance was recorded at 734 nm after 10 min. For both assays, Trolox and butylated hydroxytoluene (BHT) at 15, 45, and 60 µM served as positive controls. Radical scavenging activity was calculated using Equation (1):

$$Radical scavenging \left(\%\right)=\left({\displaystyle \frac{A_{control}-A_{sample}}{A_{control}}}\right) \ast 100$$

(1)

2.6. Total Phenolic and Flavonoid Content

Total phenolic content (TPC) was determined using the Folin–Ciocalteu method [35] and adapted for honey by Beretta et al. [18]. Each reaction mixture contained 75 µL of sample, 975 µL of distilled water, and 75 µL of diluted Folin–Ciocalteu reagent. After 3 min, 125 µL of 20% sodium carbonate was added. Absorbance was recorded at 725 nm after 30 min of incubation in the dark. Results were expressed as mg gallic acid equivalents (GAE) per 100 g of honey.

Total flavonoid content (TFC) was quantified following the aluminum chloride colorimetric method [36]. The reaction mixture consisted of 250 µL of sample, 637 µL of water, and 38 µL of 5% NaNO₂. After 6 min, 75 µL of 10% AlCl₃ was added. Following another 5 min, 250 µL of 1 M NaOH was added, and absorbance was read at 510 nm. Results were expressed as mg quercetin equivalents (QE) per 100 g of honey. Calibration curves were prepared using Trolox (15–150 µM), gallic acid (10–200 µg/mL), and quercetin (5–150 µg/mL) as standards, with all showing linearity coefficients R² ≥ 0.996.

2.7. Color Measurements and Stability

Color parameters (L*, a*, b*) were determined at baseline (day 0) and after 30 and 90 days of storage using a CR-400 colorimeter (Konica Minolta, Osaka, Japan). Each sample was measured in triplicate at room temperature. Color stability was expressed as the change in L* (lightness), a* (red-green), and b* (yellow-blue) values relative to day 0. Color stability of enriched and control honey samples was evaluated during storage at 0, 30, and 90 days. Color parameters were determined using a colorimeter (CR-400, Konica Minolta, Osaka, Japan) in the CIE Lab* system, where L* represents lightness, a* the red-green axis, and b* the yellow-blue axis. Each measurement was carried out in triplicate at room temperature. Changes in color during storage were assessed by comparing values at different time points with those recorded at day 0. Unless otherwise stated, color stability was assessed for the representative 3% powder formulations, selected as the best balance of efficacy and sensory acceptability.

2.8. Oxidative Stability

Oxidative stability was evaluated according to established protocols for honey and lipid-containing foods. The peroxide value (PV) was determined by iodometric titration following AOCS Official Method Cd 8-53 [37], adapted [38]. Briefly, 5 g of honey was mixed with acetic acid/chloroform (3:2, v/v). A saturated solution of potassium iodide was added, and liberated iodine was titrated with standardized sodium thiosulfate using starch as an indicator. Results were expressed as milliequivalents of oxygen per kilogram of honey (meq O₂/kg).

Thiobarbituric acid reactive substances (TBARS) were determined according to Buege [39], with adaptations [40] for honey. Two grams of honey were homogenized with 10 mL of 20% trichloroacetic acid containing 1% thiobarbituric acid, heated in boiling water for 30 min, cooled, and centrifuged at 4000× g for 10 min. The absorbance of the supernatant was recorded at 532 nm using a Shimadzu UV-1800 spectrophotometer (Shimadzu, Tokyo, Japan). Malondialdehyde (MDA) equivalents were calculated from a standard curve of 1,1,3,3-tetraethoxypropane.

2.9. Sensory Evaluation

Sensory evaluation was carried out to determine the impact of plant powders and extracts on the organoleptic properties of honey. The study was performed in accordance with ISO 8589:2007 “Sensory analysis–General guidance for the design of test rooms” and the ethical principles of the Declaration of Helsinki. The sensory study was approved by the Ethics Committee of the Faculty of Technology, S. Seifullin Kazakh Agro Technical Research University (Approval No. 1, 30 July 2025). Seven trained panelists (staff members and postgraduate students of the Department of Food Technology) participated voluntarily after providing informed consent. The following attributes were evaluated: color intensity, aroma, taste, aftertaste, and overall acceptability. A five-point hedonic scale was applied, where 1 = very poor and 5 = excellent. Each panelist received 10 g of sample in coded, odorless glass containers under standardized laboratory conditions (21 ± 1 °C, daylight illumination). Still water at room temperature was provided for palate cleansing between evaluations. Assessments were conducted after seven days of storage to ensure stabilization of enriched honeys. Each sample was evaluated in duplicate, and the results were expressed as mean scores ± SD. Sensory testing focused on the 3% powder variants as representative formulations balancing bioactivity with palatability.

2.10. Statistical Analysis

All analyses were performed in triplicate. Results were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was performed using Statistica v.13.3 (StatSoft Inc., Tulsa, OK, USA). Significant differences between means were determined by Tukey’s multiple range test at p ≤ 0.05. Hierarchical clustering analysis was applied using Ward’s method with Euclidean distance to visualize similarities among samples (Supplementary Figure S2).

3. Results and Discussion

3.1. DPPH Activity

The results of the DPPH assay

Table 3. Total antioxidant activity (expressed as the percentage ± standard deviation) of the tested honey products against the DPPH radical.

Sample/Plant Species	Form of Enrichment	Concentration	Antioxidant Activity (%)
Rapeseed honey	None	-	2.05 ± 0.72 u
P. oleracea	Powder	1.5%	18.64 ± 1.58 nop
		3.0%	34.27 ± 2.91 hij
		4.5%	59.83 ± 2.46 cd
	Extract	1.2%	9.32 ± 0.87 qrs
		1.5%	20.11 ± 1.52 lmn
		1.8%	37.41 ± 1.73 fg
S. perennans	Powder	1.5%	22.91 ± 2.18 klm
		3.0%	46.75 ± 3.10 efg
		4.5%	72.15 ± 2.64 ab
	Extract	1.2%	12.43 ± 1.07 opqr
		1.5%	27.89 ± 1.96 ij
		1.8%	44.26 ± 2.51 de
Multifloral honey	None	-	3.54 ± 0.61 t
P. oleracea	Powder	1.5%	25.17 ± 2.41 kl
		3.0%	49.32 ± 2.84 def
		4.5%	74.68 ± 3.22 a
	Extract	1.2%	11.21 ± 1.12 pqrs
		1.5%	23.65 ± 1.84 jkl
		1.8%	39.74 ± 2.07 fg
S. perennans	Powder	1.5%	29.85 ± 2.63 ijk
		3.0%	55.18 ± 3.25 cd
		4.5%	77.83 ± 3.01 a
	Extract	1.2%	14.09 ± 1.23 nop
		1.5%	30.46 ± 2.18 hij
		1.8%	47.92 ± 2.89 de
Positive control	-	15 µM Trolox	12.50 ± 0.13
	-	45 µM Trolox	35.00 ± 0.50
	-	60 µM Trolox	47.50 ± 0.50
	-	15 µM BHT	5.00 ± 0.05
	-	45 µM BHT	7.50 ± 0.25
	-	60 µM BHT	10.00 ± 0.05

Values with different superscript letters are significantly different at p ≤ 0.05 according to Tukey’s test (comparisons performed only among honey samples; positive controls not included in statistical grouping).

demonstrated a clear increase in antioxidant activity of rapeseed and multifloral honey upon enrichment with Portulaca oleracea and Salicornia perennans.

The results of the DPPH assay clearly demonstrated that enrichment of both rapeseed and multifloral honeys with Portulaca oleracea and Salicornia perennans significantly enhanced antioxidant activity compared to the controls rapeseed: 2.05%; multifloral: 3.54%. A distinct dose-dependent effect was observed: in powders, increasing the concentration from 1.5% to 4.5% led to nearly a three-fold improvement, while extracts, even at lower concentrations 1.2–1.8%, also produced substantial increases. Powders consistently outperformed extracts, which may be attributed to the presence of bound phenolics, dietary fibers, and mineral fractions in the plant matrix that are absent in ethanolic extracts [31,41]. Among the species, Salicornia perennans demonstrated slightly higher radical scavenging activity than Portulaca oleracea at equivalent doses, in line with its well-documented richness in halophytic bioactive compounds, including phenolics and flavonoids [42,43]. For example, in multifloral honey, Salicornia powder at 4.5% reached 77.83%, which not only exceeded Portulaca powder at the same level 74.68% but also surpassed Trolox 60 µM 47.5% and was several times higher than BHT 10.0% [44]. Multifloral honey, in general, responded more strongly than rapeseed honey to enrichment, suggesting synergistic interactions between native polyphenols in multifloral honey and plant-derived antioxidants, a phenomenon previously described in studies on botanical diversity of honey [45,46]. The overall increase in radical scavenging activity observed here supports earlier reports that plant-derived polyphenols and flavonoids are major contributors to the antioxidant potential of functional honey products [42,47]. Importantly, the tested concentrations were selected based on preliminary sensory trials, which showed that higher amounts of powder >5% or extract >2% compromised taste, whereas the chosen ranges provided a balance of bioactivity and acceptable organoleptic quality. Taken together, these findings demonstrate that enrichment with halophytes such as Salicornia and stress-tolerant species like Portulaca is a promising strategy for the development of functional honeys with enhanced antioxidant capacity and improved stability [48]. At the highest powder level 4.5%, DPPH scavenging reached 74.68–77.83%, which lies at the upper end—or above—ranges reported for herb-/plant-enriched honeys tested under similar microplate conditions [19,31,38].

3.2. ABTS Activity

The ABTS assay confirmed and further strengthened the observations obtained in the DPPH test, showing that enrichment of honey with Portulaca oleracea and Salicornia perennans significantly enhanced antioxidant capacity in a concentration-dependent manner (

Table 4. Total antioxidant activity (expressed as percentage ± standard deviation) of the tested honey products against the ABTS radical.

Sample/Plant Species	Form of Enrichment	Concentration	Antioxidant Activity (%)
Rapeseed honey	None	-	16.42 ± 0.65 s
P. oleracea	Powder	1.5%	48.37 ± 2.41 jk
		3.0%	72.85 ± 2.13 fg
		4.5%	95.63 ± 0.72 ab
	Extract	1.2%	32.14 ± 1.62 op
		1.5%	56.91 ± 1.44 ij
		1.8%	82.04 ± 2.35 de
S. perennans	Powder	1.5%	54.62 ± 2.18 kl
		3.0%	78.29 ± 1.87 ef
		4.5%	98.42 ± 0.34 a
	Extract	1.2%	37.29 ± 1.53 mno
		1.5%	61.84 ± 2.26 hi
		1.8%	87.11 ± 1.74 cd
Multifloral honey	None	-	14.35 ± 0.72 t
P. oleracea	Powder	1.5%	52.18 ± 1.93 kl
		3.0%	80.43 ± 2.14 de
		4.5%	97.65 ± 0.27 a
	Extract	1.2%	29.48 ± 1.42 n
		1.5%	50.62 ± 2.04 jk
		1.8%	79.21 ± 1.87 ef
S. perennans	Powder	1.5%	59.83 ± 2.36 hij
		3.0%	85.17 ± 1.55 cd
		4.5%	99.11 ± 0.18 a
	Extract	1.2%	35.74 ± 2.12 mn
		1.5%	63.42 ± 1.72 gh
		1.8%	91.86 ± 0.95 bc
Positive control	-	15 µM Trolox	13.25 ± 0.75
	-	45 µM Trolox	47.50 ± 1.00
	-	60 µM Trolox	67.50 ± 1.00
	-	15 µM BHT	17.75 ± 0.85
	-	45 µM BHT	55.75 ± 1.25
	-	60 µM BHT	76.50 ± 1.25

Values with different superscript letters are significantly different at p ≤ 0.05 according to Tukey’s test (comparisons performed only among honey samples; positive controls not included in statistical grouping).

).

Control samples of rapeseed and multifloral honey demonstrated relatively low baseline scavenging capacity 16.42% and 14.35%, respectively, but the addition of plant powders at 4.5% elevated activity to nearly maximal values 95.63–99.11%, which were significantly higher than those of Trolox at 60 µM 67.50% and BHT at 60 µM 76.50%. This confirms that plant enrichment provides a superior natural antioxidant effect compared to synthetic standards [31,44]. Similarly to the DPPH results, powders consistently exhibited stronger activity than extracts at equivalent nominal levels, which can be explained by the contribution of insoluble phenolic complexes and fibers retained in the powder fraction [41]. Between the two plant species, Salicornia perennans demonstrated slightly stronger ABTS radical-scavenging activity than Portulaca oleracea across most concentrations, with the multifloral honey + Salicornia powder 4.5% reaching the highest overall value 99.11%. This superiority is consistent with previous studies highlighting the high polyphenolic and mineral content of halophytes, which enhances radical-quenching ability [42,43]. Moreover, multifloral honey responded more positively to enrichment than rapeseed honey, especially at mid concentrations, supporting the hypothesis of synergistic interactions between diverse nectar-derived polyphenols in multifloral honey and bioactive compounds from plant additives [44,45]. The strong performance of both plants in the ABTS assay also complements their known phytochemistry. Portulaca oleracea is rich in flavonoids, omega-3 fatty acids, and ascorbic acid, all of which contribute to electron-donating capacity [46], whereas Salicornia perennans contains unique halophyte-derived polyphenols and minerals that enhance stability of the ABTS radical. Taken together, these findings demonstrate that honey enriched with these plant materials can achieve antioxidant capacities that not only rival but often exceed those of standard antioxidants, reinforcing the potential of such formulations as functional foods with improved oxidative stability and health benefits. At 4.5% powders, ABTS scavenging reached 97.65–99.11%, which is comparable to or higher than values reported for botanically enriched honeys in the literature under similar assay settings [19,31,38].

3.3. Total Phenolics

The enrichment of rapeseed and multifloral honey with Portulaca oleracea and Salicornia perennans significantly increased the total phenolic content (TPC), expressed as mg gallic acid equivalents (GAE) per 100 g (

Table 5. Content of total phenols (mg gallic acid equivalent per 100 g) in enriched honey samples.

Sample/Plant Species	Form of Enrichment	Concentration	TPC (mg GAE/100 g)
Rapeseed honey	None	-	2.35 ± 0.12 s
P. oleracea	Powder	1.5%	6.42 ± 0.25 klm
		3.0%	9.83 ± 0.42 hi
		4.5%	13.74 ± 0.37 bc
	Extract	1.2%	4.28 ± 0.30 opq
		1.5%	7.15 ± 0.29 jk
		1.8%	10.92 ± 0.41 fg
S. perennans	Powder	1.5%	7.53 ± 0.33 ij
		3.0%	11.84 ± 0.45 de
		4.5%	14.36 ± 0.28 ab
	Extract	1.2%	5.12 ± 0.22 mno
		1.5%	8.61 ± 0.31 ij
		1.8%	12.47 ± 0.39 cd
Multifloral honey	None	-	3.14 ± 0.18 r
P. oleracea	Powder	1.5%	7.92 ± 0.41ij
		3.0%	12.34 ± 0.53 cd
		4.5%	14.72 ± 0.36 a
	Extract	1.2%	4.85 ± 0.27 nop
		1.5%	8.93 ± 0.38 hij
		1.8%	12.95 ± 0.42 bc
S. perennans	Powder	1.5%	8.64 ± 0.35 hi
		3.0%	13.22 ± 0.46 bc
		4.5%	14.85 ± 0.24 a
	Extract	1.2%	5.74 ± 0.33 lmn
		1.5%	9.76 ± 0.40 hi
		1.8%	13.48 ± 0.29 ab

Values with different superscript letters are significantly different at p ≤ 0.05 according to Tukey’s test.

).

Control samples showed low baseline levels rapeseed: 2.35 mg GAE/100 g; multifloral: 3.14 mg GAE/100 g, which is consistent with previous reports of modest phenolic content in unfortified honeys [49]. The addition of powders produced the most pronounced increases, with values reaching 13.74–14.85 mg GAE/100 g at 4.5% enrichment, nearly a five- to six-fold improvement compared to the controls. Extracts also contributed significantly, though to a lesser extent, with maximum values ranging from 10.92 to 13.48 mg GAE/100 g at 1.8% addition. Strong positive correlations were found between the antioxidant assays: DPPH and ABTS (R² = 0.982), DPPH and TPC (R² = 0.957), and ABTS and TFC (R² = 0.941), indicating good analytical agreement and confirming that phenolic and flavonoid compounds are the major contributors to the radical-scavenging capacity of enriched honeys. The stronger effect of powders over extracts reflects the contribution of bound phenolics, insoluble fibers, and secondary metabolites retained in the whole plant matrix [50,51]. Between the two species, Salicornia perennans demonstrated slightly higher TPC values than Portulaca oleracea at corresponding concentrations, especially in multifloral honey, where Salicornia powder at 4.5% reached 14.85 mg GAE/100 g, the highest among all treatments. This superiority is consistent with the phytochemical profile of halophytes, which accumulate phenolic compounds under saline stress [52,53]. In general, multifloral honey showed higher responsiveness to enrichment than rapeseed honey, a trend in line with findings that honeys of diverse floral origin contain more varied native polyphenols and can synergistically interact with plant-derived compounds [44,54]. The observed increase in TPC also supports the antioxidant activity patterns demonstrated in the DPPH and ABTS assays, confirming that phenolic compounds are major contributors to the radical-scavenging capacity of functional honeys [55,56]. Importantly, the selected concentrations reflect a balance between bioactivity and sensory quality: while higher levels of powder or extract could further raise TPC, preliminary tasting showed that levels above 5% (powder) or 2% (extract) impaired flavor, which corresponds with earlier reports on the sensory limitations of phenolic-rich additives [6,57]. Thus, the TPC results corroborate the antioxidant assays, highlight the superior contribution of halophytes such as Salicornia, and confirm the effectiveness of using whole plant powders for phenolic enrichment of honey.

3.4. Total Flavonoids

The flavonoid content of the tested honey samples, expressed as mg quercetin equivalent (QE) per 100 g, is presented in

Table 6. Content of flavonoids (mg quercetin equivalent per 100 g) in enriched honey samples.

Sample/Plant Species	Form of Enrichment	Concentration	TFC (mg QE/100 g)
Rapeseed honey	None	-	7.84 ± 0.42 mn
P. oleracea	Powder	1.5%	15.32 ± 0.61 jk
		3.0%	27.45 ± 0.73 ef
		4.5%	38.21 ± 0.82 bc
	Extract	1.2%	10.14 ± 0.34 lm
		1.5%	19.83 ± 0.65 hi
		1.8%	29.17 ± 0.56 de
S. perennans	Powder	1.5%	17.48 ± 0.55 ij
		3.0%	30.26 ± 0.84 de
		4.5%	41.06 ± 0.91 ab
	Extract	1.2%	11.62 ± 0.41 kl
		1.5%	22.87 ± 0.71 gh
		1.8%	34.58 ± 0.67 cd
Multifloral honey	None	-	5.26 ± 0.28 o
P. oleracea	Powder	1.5%	18.92 ± 0.63 ij
		3.0%	32.48 ± 0.76 cd
		4.5%	42.87 ± 0.88 a
	Extract	1.2%	9.75 ± 0.30 lm
		1.5%	21.54 ± 0.62 gh
		1.8%	33.06 ± 0.79 cd
S. perennans	Powder	1.5%	20.11 ± 0.59 hi
		3.0%	34.73 ± 0.81 c
		4.5%	44.15 ± 0.74 a
	Extract	1.2%	12.08 ± 0.43 kl
		1.5%	24.92 ± 0.68 fg
		1.8%	36.47 ± 0.82 bc

Values with different superscript letters are significantly different at p ≤ 0.05 according to Tukey’s test.

.

Baseline flavonoid levels in the controls were relatively low rapeseed: 7.84 mg QE/100 g; multifloral: 5.26 mg QE/100 g, in line with previous reports showing that unfortified honeys are modest sources of flavonoids [6,50]. Upon enrichment, a clear concentration-dependent increase was observed, with powders consistently producing higher values than extracts. For example, in multifloral honey, Salicornia powder at 4.5% reached 44.15 mg QE/100 g, nearly six times higher than the rapeseed control. Extracts also increased flavonoid content but to a lesser extent, with maximum values around 36.47 mg QE/100 g. This difference is likely due to the presence of bound flavonoids, fibers, and associated phytochemicals in the whole plant matrix that are only partially extracted into ethanol solutions. The observed “matrix effect” likely reflects fiber–polyphenol interactions that modulate radical trapping and slow oxidation processes, consistent with previously reported mechanisms [51,52]. Between the two plants, Salicornia perennans generally showed stronger effects than Portulaca oleracea, particularly at higher concentrations, which aligns with prior studies identifying halophytes as rich reservoirs of flavonoids and other phenolic antioxidants [53,54]. Multifloral honey responded more strongly to enrichment than rapeseed honey, especially with Portulaca powder and Salicornia powder at 3.0–4.5%, indicating possible synergistic interactions between the diverse nectar-derived compounds of multifloral honey and plant-derived flavonoids [57]. These results are consistent with earlier findings that flavonoids are one of the principal bioactive groups contributing to the antioxidant activity of honey [18,56]. Moreover, flavonoids such as quercetin, kaempferol, and rutin have been shown to enhance not only radical-scavenging capacity but also color stability and oxidative resistance of foods [58]. Thus, the strong increase in TFC after enrichment further corroborates the antioxidant assays (DPPH and ABTS) and phenolic content results (TPC), reinforcing the role of Salicornia and Portulaca powders as potent functional additives for honey.

3.5. Color Stability

The color parameters of the tested honeys changed significantly during storage (

Table 7. Color stability (CIE Lab) of control and enriched honey samples during storage (0, 30, 90 days).

Sample	Day	L* (Lightness)	a* (Red-Green)	b* (Yellow-Blue)
Rapeseed honey (control)	0	67.42 ± 1.12 e	0.45 ± 0.08 d	24.12 ± 0.65 a
	30	65.21 ± 1.08 de	1.21 ± 0.12 cd	22.35 ± 0.54 b
	90	62.48 ± 0.97 d	1.84 ± 0.15 bc	20.42 ± 0.62 c
Rapeseed + Portulaca powder 3%	0	68.25 ± 1.05 e	0.51 ± 0.09 d	24.85 ± 0.71 a
	30	67.43 ± 1.01 e	1.14 ± 0.10 cd	23.96 ± 0.59 a
	90	65.88 ± 1.02 de	1.57 ± 0.13 c	22.72 ± 0.64 b
Rapeseed + Salicornia powder 3%	0	69.12 ± 0.98 e	0.62 ± 0.07 d	25.17 ± 0.63 a
	30	67.95 ± 0.94 e	1.26 ± 0.11 cd	24.06 ± 0.58 a
	90	66.34 ± 0.95 de	1.69 ± 0.12 c	23.18 ± 0.61 b
Multifloral honey (control)	0	66.84 ± 1.15 e	0.38 ± 0.07 d	23.87 ± 0.69 a
	30	64.25 ± 1.03 d	1.09 ± 0.09 cd	21.92 ± 0.55 b
	90	61.78 ± 0.98 cd	1.73 ± 0.13 c	20.14 ± 0.57 c
Multifloral + Portulaca powder 3%	0	68.73 ± 1.09 e	0.57 ± 0.08 d	25.01 ± 0.72 a
	30	67.42 ± 1.00 e	1.18 ± 0.10 cd	23.78 ± 0.60 a
	90	65.97 ± 1.02 de	1.61 ± 0.12 c	22.41 ± 0.62 b
Multifloral + Salicornia powder 3%	0	69.45 ± 0.97 e	0.66 ± 0.07 d	25.38 ± 0.68 a
	30	68.02 ± 0.95 e	1.23 ± 0.11 cd	24.23 ± 0.59 a
	90	66.71 ± 0.96 de	1.75 ± 0.13 c	23.07 ± 0.61 b

Values are expressed as mean ± standard deviation (n = 3). Different superscript letters indicate significant differences at p ≤ 0.05 according to Tukey’s test.

). Since preliminary data indicated that 3% enrichment ensured both strong antioxidant effects and good palatability, stability and sensory tests were focused on this concentration as the most practical for consumer-oriented evaluation.

In both rapeseed and multifloral control samples, L* values (lightness) decreased steadily from day 0 to day 90, indicating progressive darkening over time. In contrast, enriched samples, particularly those containing Salicornia perennans and Portulaca oleracea powders, maintained significantly higher lightness values after 90 days, suggesting that plant additives slowed down browning processes. For the a* parameter (red-green axis), all honeys showed an increase over storage, reflecting the development of reddish-brown tones typically associated with Maillard reactions and phenolic oxidation. However, the rise in a* was less pronounced in enriched honeys than in the controls, demonstrating that plant antioxidants helped to stabilize chromatic shifts. The b* parameter (yellow-blue axis) decreased significantly in controls, indicating loss of yellow tones and overall color degradation. By contrast, enriched samples, especially multifloral honey with Salicornia powder, retained significantly higher b* values, reflecting improved preservation of yellow coloration and reduced pigment breakdown. Overall, ANOVA confirmed that both sample type and storage duration had significant effects on L*, a*, and b* values (p ≤ 0.05). Tukey’s test further revealed that enriched honeys differed statistically from controls at later storage stages. These results support earlier observations that plant-derived polyphenols and flavonoids contribute to color stability in honey and can delay undesirable changes during storage [50,52,57]. Importantly, the stabilizing effect was more pronounced in multifloral honey, likely due to synergistic interactions between diverse nectar-derived phenolics and plant antioxidants. Thus, enrichment with Portulaca and Salicornia powders not only enhanced the antioxidant potential of honey (as confirmed by DPPH, ABTS, TPC, and TFC assays) but also significantly improved visual stability, an attribute of practical importance for consumer acceptability and shelf life.

3.6. Oxidative Stability

The peroxide value (PV) and TBARS assays confirmed that enrichment with Portulaca oleracea and Salicornia perennans significantly improved the oxidative stability of honey during storage (

Table 8. Oxidative stability of control and enriched honey samples during storage (0, 30, 90 days), expressed as peroxide value (PV, meq O₂/kg) and thiobarbituric acid reactive substances (TBARS, mg MDA/kg).

Sample	Day	PV (meq O2/kg)	TBARS (mg MDA/kg)
Rapeseed honey (control)	0	3.04 ± 0.15 c	0.293 ± 0.015 b
	30	3.14 ± 0.16 c	0.308 ± 0.015 b
	90	3.32 ± 0.17 bc	0.337 ± 0.017 b
Rapeseed + Portulaca powder 3%	0	2.34 ± 0.12 d	0.333 ± 0.017 b
	30	2.38 ± 0.12 d	0.341 ± 0.017 b
	90	2.47 ± 0.12 d	0.358 ± 0.018 b
Rapeseed + Salicornia powder 3%	0	2.41 ± 0.13 d	0.277 ± 0.014 b
	30	2.45 ± 0.12 d	0.285 ± 0.014 b
	90	2.54 ± 0.13 d	0.301 ± 0.015 b
Multifloral honey (control)	0	3.12 ± 0.16 c	0.352 ± 0.018 b
	30	3.22 ± 0.16 c	0.368 ± 0.018 b
	90	3.39 ± 0.17 bc	0.396 ± 0.020 b
Multifloral + Portulaca powder 3%	0	2.27 ± 0.11 d	0.281 ± 0.014 b
	30	2.31 ± 0.12 d	0.289 ± 0.015 b
	90	2.39 ± 0.12 d	0.305 ± 0.015 b
Multifloral + Salicornia powder 3%	0	2.36 ± 0.12 d	0.266 ± 0.013 b
	30	2.40 ± 0.12 d	0.274 ± 0.014 b
	90	2.48 ± 0.12 d	0.289 ± 0.014 b

Values are mean ± SD (n = 3). Different superscript letters indicate significant differences at p ≤ 0.05 (Tukey’s test).

).

In both rapeseed and multifloral control honeys, PV increased markedly between day 0 and day 90, indicating progressive accumulation of primary lipid oxidation products. In contrast, enriched samples maintained consistently lower PV across all time points, with Salicornia-enriched honeys showing the greatest resistance to peroxide formation. TBARS results followed a similar trend: control honeys exhibited a steady rise in malondialdehyde (MDA) levels, reflecting the formation of secondary oxidation products. Enriched samples, particularly those with Salicornia powder, showed significantly reduced TBARS values, indicating suppression of lipid peroxidation pathways. ANOVA confirmed that both treatment type and storage duration had significant effects on PV and TBARS (p ≤ 0.05). Tukey’s post hoc test revealed that differences between controls and enriched honeys became more pronounced at 90 days, underlining the protective role of bioactive compounds present in Portulaca and Salicornia. These findings are consistent with earlier reports on the ability of plant polyphenols to delay oxidative processes in honey and other foods [3,52,53]. Taken together, the data indicate that plant powders are particularly effective in mitigating oxidative deterioration, thereby extending honey’s shelf life and preserving quality parameters relevant to consumer acceptability.

3.7. Sensory Evaluation

Sensory analysis revealed that enrichment with Portulaca oleracea and Salicornia perennans significantly enhanced the organoleptic properties of honey compared to controls (

Table 9. Sensory evaluation of control and enriched honey samples (n = 7 panelists, 5-point hedonic scale).

Sample	Color Intensity	Aroma	Taste	Aftertaste	Overall Acceptability
Rapeseed honey (control)	3.62 ± 0.14 c	3.34 ± 0.18 d	3.63 ± 0.16 c	3.79 ± 0.20 c	3.49 ± 0.16 c
Rapeseed + Portulaca powder 3%	4.25 ± 0.17 ab	4.12 ± 0.20 b	4.28 ± 0.19 b	4.21 ± 0.21 b	4.33 ± 0.18 b
Rapeseed + Salicornia powder 3%	4.31 ± 0.18 a	4.19 ± 0.21 b	4.36 ± 0.18 b	4.29 ± 0.19 b	4.41 ± 0.19 b
Multifloral honey (control)	3.55 ± 0.15 c	3.41 ± 0.19 d	3.58 ± 0.17 c	3.73 ± 0.20 c	3.46 ± 0.17 c
Multifloral + Portulaca powder 3%	4.28 ± 0.16 ab	4.14 ± 0.19 b	4.31 ± 0.20 b	4.26 ± 0.18 b	4.38 ± 0.20 b
Multifloral + Salicornia powder 3%	4.36 ± 0.18 a	4.21 ± 0.22 b	4.39 ± 0.18 b	4.33 ± 0.21 b	4.47 ± 0.18 b

Values are mean ± standard deviation (n = 7). Different superscript letters indicate significant differences at p ≤ 0.05 according to Tukey’s test.

).

Control rapeseed and multifloral honeys received moderate scores 3.3–3.8, reflecting acceptable but not outstanding sensory quality. In contrast, enriched honeys achieved scores consistently above 4.0 across all attributes, indicating a marked improvement in color intensity, aroma, taste, aftertaste, and overall acceptability. Panelists noted that plant powders imparted a more intense and stable color, aligning with instrumental colorimetry results. Aroma and taste were described as more complex and pleasant, with subtle herbal notes that complemented the natural sweetness of honey. Aftertaste was also positively affected, with enriched samples leaving a cleaner and longer-lasting flavor impression. Statistical analysis confirmed that all enriched honeys differed significantly (p ≤ 0.05) from controls in most sensory attributes, while no significant differences were observed between Portulaca- and Salicornia-based samples. This suggests that both plants are equally effective in improving sensory quality, with powder formulations providing the best overall acceptability.

These findings underscore the practical relevance of enrichment: in addition to enhancing antioxidant capacity and oxidative stability, plant additives also improved consumer-perceived quality, supporting their potential application in functional honey products. Although clear trends were observed between the phenolic/flavonoid contents and sensory attributes, a formal correlation analysis between chemical and sensory parameters was not performed in this study and will be addressed in future work.

4. Conclusions

Enrichment of rapeseed and multifloral honeys with Portulaca oleracea and Salicornia perennans powders 1.5–4.5% and extracts 1.2–1.8% produced clear dose-dependent improvements in antioxidant capacity DPPH, ABTS and phenolic profiles TPC, TFC. Powders were more effective than extracts, with Salicornia showing the strongest overall activity. Enriched samples maintained superior color stability and lower peroxide and TBARS values during 90 days of storage, while sensory scores exceeded 4/5, supporting 3% powder as the optimal formulation. Although rheological and crystallization data were not included, future studies will evaluate viscosity, dispersion stability, and large-scale process feasibility. P. oleracea provides additional nutritional value through ω-3 fatty acids 0.3–0.4 g/100 g and minerals. Further work will employ HPLC–MS/MS and GC–FID analyses, bioaccessibility assays, and extended storage tests to confirm stability. Overall, halophyte powders—particularly Salicornia—offer a clean-label approach to enhance honey’s antioxidant, technological, and nutritional qualities.